PROJECT SUMMARY Cell membranes are the fabric of life. Membrane structure, dynamics, and function are influenced by phospholipid composition. The phospholipid milieu can influence the structure, dynamics, and function of proteins such as transporters and receptors. Maintaining and regulating the abundance of hundreds of phospholipid species at appropriate ratios involves a host of cellular mechanisms. Some of these mechanisms operate within and among the enzymes that synthesize and remodel phospholipids. We are focused on understanding the mechanisms that regulate the allocation of acyl chains among phospholipid precursors. Such knowledge may facilitate therapies to influence protein function via influencing membrane structure, to address the phospholipidosis associated with cationic amphiphilic drugs, and to attenuate pro-inflammatory signals that the phospholipid precursors 1-acylglycerol-3-phosphate (lysoPA) and phosphatidate can send. Recently, we found that compound deletion of different acyltransferases in Saccharomyces cerevisiae caused a gradation of phospholipid composition phenotypes. Algebraic analysis of this in vivo data suggested that the two major 1-acylglycerol-3-phosphate O-acyltransferases (AGPAT), which have two substrates containing acyl chains, uniquely pair substrates based on the respective acyl chain lengths. We propose to perform exhaustive, in vitro assays to determine kinetic parameters for these two AGPATs, Slc1 and Lpt1, using a two- by-two array of lysoPA and acyl-CoA substrate pairings. If evidence of selective pairing is found, the three closest human homologs for the respective yeast AGPATs will be expressed in Sf9 insect cells. Microsomes from these cells will undergo the same two-by-two array of substrate pairings. This may establish a novel mechanism for regulating phospholipid composition in human cells. Secondly, we will take a broader approach and test the hypothesis there is substrate channeling among the reactions that sequentially incorporate fatty acids into CDP-DAG. CDP-DAG is the phospholipid precursor onto which head groups are attached. Even in the relatively simple metabolic framework of S. cerevisiae, each reaction between fatty acids and CDP-DAG is mediated by multiple isoenzymes. We will use the membrane yeast two-hybrid assay to test for the 148 possible physical interactions among the 22 enzymes and binding proteins that mediate the sequential yet branched reactions between fatty acids and CDP-DAG. If specific interactions indicating channeling are found, the respective, human homologs will be similarly assayed. Homeostatic mechanisms that regulate phospholipid composition may do so via transcript abundance. To identify novel mechanisms, yeast strains with the four compound gene-deletion genotypes with a gradation of phospholipid phenotypes will undergo RNA sequencing. Statistical analysis will be developed to identify specific or clustered transcripts proportionately altered by single or clustered phospholipid species. Parallel studies will first genetically remove the Opi1 transcription factor known to regulate phospholipid production.